phosphorus-radioisotopes and dehydroretronecine

Riddelliine is a naturally occurring carcinogenic pyrrolizidine alkaloid that produces liver tumors in experimental animals. Riddelliine requires metabolic activation to dehydroriddelliine and 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) to exert its toxicity. Previously, (32)P-postlabeling HPLC was used to detect a set of eight DHP-derived adduct peaks from DNA modified both in vitro and in vivo. Among these DHP-derived DNA adducts, two were identified as epimers of DHP-2'-deoxyguanosine 3'-monophosphate. In this study, the remaining adducts have been characterized as DHP-modified dinucleotides. A series of dinucleotides, TpGp, ApGp, TpCp, ApCp, TpAp, ApAp, TpTp, and ApTp, were obtained by enzymatic digestion of calf thymus DNA with micrococcal nuclease (MN) and spleen phosphodiesterase (SPD) followed by HPLC separation and structural identification by negative ion electrospray tandem mass spectrometry (ES/MS/MS). Incubation of individual dinucleotides with DHP produced DHP-modified dinucleotide adducts that were also characterized using LC-ES/MS/MS. A parallel analysis of the isolated DHP-modified dinucleotides using (32)P-postlabeling recapitulated the series of unidentified adduct peaks that we previously reported from DHP-modified calf thymus DNA in vitro and rat liver DNA in vivo. Intact calf thymus DNA was also reacted with DHP and then digested by MN/SPD under the same conditions. The adduct profile obtained from LC-ES/MS/MS analysis was similar to that observed from the isolated dinucleotides. Structural analysis using LC-ES/MS/MS showed that DHP bound covalently to both 3'- and 5'-guanine, -adenine, and -thymine bases (but not cytosine) of dinucleotides to produce two or more isomers of each DHP-dinucleotide adduct. By comparing adduct formation at dissimilar bases within individual dinucleotides, the relative reactivity of DHP with individual bases was determined to be guanine > adenine approximately thymine. Identification of the entire set of DHP-derived DNA adducts further validates the conclusion that riddelliine is a genotoxic carcinogen and enhances the applicability of these biomarkers for assessing carcinogenic risks from exposure to pyrrolizidine alkaloids.

Topics: Adenine Nucleotides; Administration, Oral; Animals; Carcinogens; Cattle; DNA; DNA Adducts; Guanine Nucleotides; Isomerism; Liver; Micrococcal Nuclease; Monocrotaline; Phosphorus Radioisotopes; Pyrrolizidine Alkaloids; Rats; Spectrometry, Mass, Electrospray Ionization; Spleen; Thymine Nucleotides; Thymus Gland

Pyrrolizidine alkaloids are naturally occurring genotoxic chemicals produced by a large number of plants. Metabolism of pyrrolizidine alkaloids in vivo and in vitro generates dehydroretronecine (DHR) as a common reactive metabolite. In this study, we report the development of a (32)P-postlabeling/HPLC method for detection of (i) two DHR-3'-dGMP and four DHR-3'-dAMP adducts and (ii) a set of eight DHR-derived DNA adducts in vitro and in vivo. The approach involves (1) synthesis of DHR-3'-dGMP, DHR-3'-dAMP, and DHR-3',5'-dG-bisphosphate standards and characterization of their structures by mass and (1)H NMR spectral analyses, (2) development of optimal conditions for enzymatic DNA digestion, adduct enrichment, and (32)P-postlabeling, and (3) development of optimal HPLC conditions. Using this methodology, we have detected eight DHR-derived DNA adducts, including the two epimeric DHR-3',5'-dG-bisphosphate adducts both in vitro and in vivo.

Topics: Animals; Carcinogens; Cattle; Chromatography, High Pressure Liquid; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA; DNA Adducts; Exonucleases; Female; Isotope Labeling; Micrococcal Nuclease; Monocrotaline; Phosphorus Radioisotopes; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344; Reproducibility of Results; Single-Strand Specific DNA and RNA Endonucleases; Spectrometry, Mass, Electrospray Ionization

Riddelliine is a representative naturally occurring genotoxic pyrrolizidine alkaloid. We have studied the mechanism by which riddelliine induces hepatocellular tumors in vivo. Metabolism of riddelliine by liver microsomes of F344 female rats generated riddelliine N-oxide and dehydroretronecine (DHR) as major metabolites. Metabolism was enhanced when liver microsomes from phenobarbital-treated rats were used. Metabolism in the presence of calf thymus DNA resulted in eight DNA adducts that were identical to those obtained from the reaction of DHR with calf thymus DNA. Two of these adducts were identified as DHR-modified 7-deoxyguanosin-N(2)-yl epimers (DHR-3'-dGMP); the other six were DHR-derived DNA adducts, but their structures were not characterized. A similar DNA adduct profile was detected in the livers of female F344 rats fed riddelliine, and a dose-response relationship was obtained for the level of the total (eight) DHR-derived DNA adducts and the level of the DHR-3'-dGMP adducts. These results suggest that riddelliine induces liver tumors in rats through a genotoxic mechanism and the eight DHR-derived DNA adducts are likely to contribute to liver tumor development.

Topics: Animals; Biotransformation; Carcinogens; Cattle; Chromatography, High Pressure Liquid; DNA; DNA Adducts; Female; Isotope Labeling; Liver; Liver Neoplasms, Experimental; Microsomes, Liver; Monocrotaline; Phosphorus Radioisotopes; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344